Lax Alistair J. Bacterial protein toxins: Role in the interference with cell growth regulation (Бактериальные токсины белков: роль в регуляции роста клеток)

Подождите немного. Документ загружается.

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Figure 2.5. Proliferation of the bladder epithelium of pigs infected with toxigenic

P. multocida.A,normal bladder epithelium; B, epithelium from pig infected with

toxigenic P. multocida.

mostly non-toxigenic. However, those from respiratory infections likely to

have been acquired from pigs, for example from farm workers, are sometimes

toxigenic (Donnio et al., 1991). Their long-term carriage could have serious

implications.

CONCLUSIONS

The Pasteurella multocida toxin is a highly unusual toxin that is mitogenic

for many cell types. Most bacterial toxins are associated with acute diseases

in which cells are killed or their function is severely impaired. Although

PMT has long been associated with the pig disease atrophic rhinitis, chronic

exposure to the toxin may also have hidden consequences as its effects on

cell signalling show many similarities with those of tumour promoters. Very

few studies have looked at this possibility, although it is now widely accepted

that bacteria can cause cancer; in particular, Helicobacter pylori is linked with

gastric cancer (see Chapter 8). This is an important area for future work on

this toxin.

PMT is an activator of G

-mediated signalling pathways, and possibly

also of the G

class of G-proteins. It is the first toxin known to be an agonist

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the

pasteurella multocida

toxin

for G

. Toxins that specifically activate or inactivate other heterotrimeric G-

proteins, for example cholera toxin and pertussis toxin, have been widely

used as reagents to help unravel cell-signalling pathways. It is anticipated that

PMT will also become an increasingly valuable tool for cell biology research,

particularly after its molecular mode of action is fully understood.

ACKNOWLEDGEMENT

I thank the Wellcome Trust for its support.

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CHAPTER 3

Rho-activating toxins and growth regulation

Gudula Schmidt and Klaus Aktories

Low molecular mass GTPases of the Rho family are master regulators of the

actin cytoskeleton and are involved in various signal transduction processes.

They regulate the actin cytoskeleton and control transcriptional activation,

cell cycle progression, cell transformation, and apoptosis. Rho GTPases are

the eukaryotic targets of a variety of bacterial protein toxins that either in-

hibit or activate the Rho proteins. The cytotoxic necrotizing factors CNF1

and 2 from Escherichia coli and the dermonecrotic toxin DNT from Borde-

tella species are Rho-activating toxins. Whereas CNFs cause deamidation of

Rho proteins, DNT deamidates and/or transglutaminates the GTPases by

attachment of polyamines. Both deamidation and transglutamination cause

persistent activation of Rho GTPases and subsequent changes in processes

governed by the GTPases.

RHOGTPASES

Rho GTPases belong to the Ras superfamily of low molecular mass GTPases

and are molecular switches in various signalling pathways. Like all members

of GTP-binding proteins, they are active in the GTP bound form and inac-

tive with GDP bound. Activation occurs by GDP/GTP exchange catalysed by

guanine nucleotide exchange factors (GEFs) and is inhibited by binding of

guanine nucleotide dissociation inhibitors (GDIs). Inactivation by hydrolysis

of the bound GTP is catalysed by GTPase activating proteins (GAPs) (Symons

and Settleman, 2000).

Rho GTPases are ubiquitously expressed. The members (>15) of the Rho

family of GTPases, including RhoA, B, C, D, E, and G, Cdc42, Rac1, 2, and 3,

share more than 50% sequence identity. The GTPases have been described as

important regulators of the actin cytoskeleton. RhoA regulates the formation

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gudula schmidt and klaus aktories

Pak

IRSp53

profilin

gelsolin

PI5K

PIP2

LimK

cofilin

WAVE’s

Arp2/3

Rac

relaxation of

contractile

forces

actin

polymerization

lamellipodia

actin

filament

stabilisation

Pak

IRSp53

profilin

gelsolin

PI5K

PIP2

LimK

cofilin

WAVE’s

Arp2/3

Rac

relaxation of

contractile

forces

actin

polymerization

lamellipodia

actin

filament

stabilisation

Pak

IRSp53

profilin

gelsolin

PI5K

PIP2

LimK

cofilin

WAVE’s

Arp2/3

Rac

relaxation of

contractile

forces

actin

polymerization

lamellipodia

actin

filament

stabilisation

Arp2/3

WASP’s

profilin

G-actin

Cdc42

actin

polymerization

filopodia

mDia

profilin

G-actin

Rock

MLC

MLC-P Arp2/3

MLC-

Ptase

MLC-

kinase

RhoA

increased

actin

polymerization

actomyosin

assembly

and contraction

mDia

profilin

G-actin

Rock

MLC

MLC-P Arp2/3

MLC-

Ptase

MLC-

kinase

RhoA

increased

actin

polymerization

actomyosin

assembly

and contraction

mDia

profilin

G-actin

Rock

MLC

MLC-P Arp2/3

MLC-

Ptase

MLC-

kinase

RhoA

increased

actin

polymerization

actomyosin

assembly

and contraction

Phagocytosis, cell spreading and migration

stress fibers, focal adhesions and cell motility

LimK

cofilin

Figure 3.1. Rho GTPases regulate the actin architecture by different pathways. Cdc42,

Rac, and RhoA activate the Arp2/3 complex via different effector molecules, which are

protein kinases (e.g., PI5K, PAK, ROCK) or scaffolding molecules (e.g., WASP, IRS p53,

mDia). These effector molecules also regulate other signalling pathways, leading to actin

polymerisation or depolymerisation and, thus, control cellular morphology, movement,

and other actin-dependent processes.

of actin stress fibres – bundles of actin filaments associated with myosin fila-

ments and other proteins. Cdc42 is known to induce formation of filopodia,

which are small actin protrusions. Similar to stress fibres, filopodia are asso-

ciated with focal contacts, which link the cytoskeleton with the extracellular

matrix and the substratum. It has been suggested that filopodia are sensory

elements more than the driving force for migrating cells. The third well-

characterised member of the Rho family, Rac, is involved in the formation

of lamellipodia and membrane ruffles. These actin-containing structures are

necessary for phagocytosis and cell movement. By regulating the actin cy-

toskeleton, Rho-GTPases are involved in migration, phagocytosis, endo- and

exocytosis, and cell–cell and cell–matrix contact (Nobes and Hall, 1999).

Furthermore, the family of Rho GTPases is involved in the regulation

of transcriptional activation, cell cycle progression, cell transformation, and

apoptosis. Many Rho GTPase effectors involved in these processes have been

identified, including protein- and lipid kinases, phospholipase D, scaffolding

proteins, and numerous adaptor proteins (Bishop and Hall, 2000).

The pathways leading to actin-based motility are the best characterised

(Figure 3.1). RhoA-dependent reorganisation of the actin cytoskeleton is

mediated by the cooperative action of Rho kinase (p160/ROCK) (Rho-

associated kinase) and mDia (diaphanous) proteins. mDia proteins interact

with actin via their C-terminal FH2 domain (Alberts, 2001) and regulate the

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toxins that activate rho

profilin–G-actin interaction via their FH1 domain, thereby leading to actin

polymerisation. Rho kinase, on the other hand, increases the myosin light

chain (MLC) phosphorylation by inhibiting the MLC phosphatase and,

thereby, facilitates the assembly of actin and myosin (Essler et al., 1998). More-

over, Rho-kinase, and also protein kinase C (PKC), phosphorylates calponin

and, thereby, reduces calponin–actin interaction (Kaneko et al., 2000). Bind-

ing of calponin to actin has an inhibitory effect on the ATPase activity of

myosin (for review see Fukata et al., 2001). In some cells, Rac antagonises

the action of RhoA by inhibiting the MLC kinase, myosin phosphorylation,

and the actomyosin assembly. Inactivation of the MLC kinase by Rac in-

volves PAK, an effector of Rac and Cdc42. Besides inhibiting contractile

forces, PAK leads to the stabilisation of actin filaments by activating LIM

kinase, which inhibits cofilin by phosphorylation. Cofilin severs actin fila-

ments and additionally increases the rate of monomer release at the pointed

ends of actin filaments. However, Rho kinase also activates Lim kinase to act

in this case synergistically with Rac signalling pathways. This effect might

be of importance for RhoA-induced actin polymerisation. Activated Rac and

Cdc42 stimulate actin nucleation by the Arp2/3 complex. This involves WASP

(Wiskott-Aldrich syndrome protein) family proteins. Whereas WASP and

the ubiquitously expressed N-WASP proteins are activated by Cdc42, WAVE

(WASP verproline homologous protein) is regulated by Rac.

RHOGTPASES AS REGULATORS OF PROLIFERATION

The precise pathways whereby Rho proteins impinge on the regulation of

proliferation are not particularly clear. In mammalian cells, proliferation is

cooperatively controlled by soluble growth factors and cell adhesion to the

extracellular matrix. Signalling pathways induced by both stimuli cross-talk

on several regulatory levels and are regulated by various Rho GTPases. These

include regulation of integrin signalling, control of MAP kinase cascades sub-

sequent to tyrosine kinase receptor activation, and activation of PI3kinase-

dependent pathways (Assoian and Schwartz, 2001; Danen and Yamada,

2001). For example, mitogenic signals from soluble growth factors are trans-

duced by the canonical Ras-Raf-MEK-ERK cascade with subsequent tran-

scriptional activation. Integrin signalling converges via focal adhesion kinase

(p125

FAK

), paxillin, and src kinases with MAP kinase cascade regulation. The

functional consequences are transcriptional activation (e.g., increase in cy-

clin D1 transcription) and cell cycle progression, eventually resulting in cell

growth.